Sequential enzyme delivery system

ABSTRACT

A process for fabric washing in which fabric is treated sequentially with at least two different enzymes, the process comprising the steps: 1. treatment with one or more proteases, followed by 2. treatment with one or more of a second enzyme characterised in that the or each second enzyme(s) of the second step is/are of a different enzyme family to the protease(s) in the first step.

The present invention concerns the sequential delivery of enzymes in a washing process.

The of enzyme mixtures during a laundering process is known. WO2000070006 discloses protease/cellulose combinations. WO20050089966, EP1664258, US20050059567 and US20060172913 disclose enzyme mixtures for grass and egg stain removal.

An objective is to provide an improved washing/stain removal process involving enzymes. It is also very difficult to provide a blend of enzymes whereby protease is one of those major components due to hydrolysis of the other components.

Accordingly, in a first aspect, the present invention provides a fabric washing process in which fabric is treated sequentially with at least two different enzymes, the process comprising the steps:

1. treatment with one or more proteases, followed by 2. treatment with one or more of a second enzyme, characterised in that the or each second enzyme(s) of the second step is/are of a different family to the proteases of the first step.

Preferably the second enzyme(s) comprise one or more lipolytic enzymes

In a second aspect the invention provides a process for removing stains from fabric comprising the steps:

1. treatment with one or more proteases, followed by 2. treatment with one or more of a second enzyme, characterised in that the or each second enzyme(s) of the second step is/are being of a different family to the proteases of the first step.

Preferred features of the process of the second aspect are as for the first aspect.

There may be a time delay between the steps or another process step such as a rinse step.

Preferably the process of the second aspect of the invention is a process for removing grass stains from fabric

In another aspect the invention provides a fabric washing and/or stain removal kit including at least one package containing first and second enzymes according to the first aspect which are separated from each other, the package optionally including instructions for washing fabric and/or removing stains from fabric according to the first and/or second aspects of the invention.

In a further aspect the invention provides a washing machine incorporating a device for sequentially treating fabrics with at least first and second enzymes according to the first aspect, the device comprising a plurality of separate chambers containing respectively first and second enzymes, from which chambers the enzymes are sequentially dispensed. The device preferably comprises the drawer of a washing machine.

In a further aspect, the invention provides the use sequentially of first then second enzymes according to the first aspect in a process for treatment of stains, particularly grass stains from fabric.

The first and second enzymes may comprise a single enzyme or a mixture of enzymes.

Surprisingly, the above arrangement allows the enzymes to work with minimal interference from other enzymes. The proteases, dosed first, might be expected to attack the other enzyme(s) on addition to the wash liquor preventing the non proteolytic treatment of stains. However, the stain removal performance of the enzymes due to sequential dosing is vastly improved.

Suitable proteases include those of animal, vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered mutants are included. The protease may be a serine protease or a metallo protease, preferably an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases are subtilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279). Examples of trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described in WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants with substitutions in one or more of the following positions: 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and 274. Preferred commercially available protease enzymes include Alcalase™, Savinase™, Primase™, Duralase™, Dyrazym™, Esperase™, Everlase™, Polarzyme™, and Kannase™, (Novozymes A/S), Maxatase™, Maxacal™, Maxapem™, Properase™, Purafect™, Purafect OxP™, FN2™, and FN3™ (Genencor International Inc.).

Suitable lipases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful lipases include lipases from Humicola (synonym Thermomyces), e.g. from H. lanuginosa (T. lanuginosus) as described in EP 258 068 and EP 305 216 or from H. insolens as described in WO 96/13580, a Pseudomonas lipase, e.g. from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P. fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g. from B. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta, 1131, 253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422).

Other examples are lipase variants such as those described in WO 92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202.

Preferred commercially available lipase enzymes include Lipolase™ and Lipolase Ultra™, Lipex™ (Novozymes A/S). It is to be understood that enzyme variants (produced, for example, by recombinant techniques) are included within the meaning of the term “enzyme”. Examples of such enzyme variants are disclosed, e.g., in EP 251,446 (Genencor), WO 91/00345 (Novo Nordisk), EP 525,610 (Solvay) and WO 94/02618 (Gist-Brocades NV).

Accordingly the types of enzymes which may appropriately be incorporated in granules of the invention include oxidoreductases (EC 1.-.-.-), transferases (EC 2.-.-.-), hydrolases (EC 3.-.-.-), lyases (EC 4.-.-.-), isomerases (EC 5.-.-.-) and ligases (EC 6.-.-.-).

The sequentially delivered proteases and lipolytic enzymes may be dosed in isolation from other main wash components or they may be dosed with such components, but preferably not combining the protease with other enzymes.

The sequentially delivered enzymes of the invention may be delivered together with one or more surfactants and/or optionally other ingredients such that at least one of the sequential doses is a fully functional laundry cleaning and/or care compositions. Such compositions of the invention may be in dry solid or liquid form. The composition may be a concentrate to be diluted, rehydrated and/or dissolved in a solvent, including water, before use. The composition may also be a ready-to-use (in-use) composition.

The present invention is suitable for use in industrial or domestic fabric wash compositions, fabric conditioning compositions and compositions for both washing and conditioning fabrics (so-called through the wash conditioner compositions). The present invention can also be applied to industrial or domestic non-detergent based fabric care compositions, for example spray-on compositions.

Fabric wash compositions according to the present invention may be in any suitable form, for example powdered, tableted powders, liquid or solid detergent bars.

Other contemplated ingredients including surfactants, hydrotropes, preservatives, fillers, builders, complexing agents, polymers, stabilizers, perfumes per se, other detergent ingredients, or combinations of one or more thereof are discussed below.

The enzymes may be present as the sole reactive stain removal agent, or other stain removal agents may be incorporated.

Additional enzymes may be dosed as part of the overall washing process, providing this follows the initial process according to the invention (i.e. treatment with an enzyme such as a protease/s and then a second or more enzyme such as lipase/s).

Such additional (i.e. subsequently dosed) enzymes may include further proteases and lipases as above, and also alpha-amylases, cellulases, peroxidases/oxidases, pectate lyases, and mannanases, or mixtures thereof.

Additional components may also include cutinase. classified in EC 3.1.1.74. The cutinase used according to the invention may be of any origin. Preferably cutinases are of microbial origin, in particular of bacterial, of fungal or of yeast origin.

Cutinases are enzymes which are able to degrade cutin. In a preferred embodiment, the cutinase is derived from a strain of Aspergillus, in particular Aspergillus oryzae, a strain of Alternaria, in particular Alternaria brassiciola, a strain of Fusarium, in particular Fusarium solani, Fusarium solani pisi, Fusarium roseum culmorum, or Fusarium roseum sambucium, a strain of Helminthosporum, in particular Helminthosporum sativum, a strain of Humicola, in particular Humicola insolens, a strain of Pseudomonas, in particular Pseudomonas mendocina, or Pseudomonas putida, a strain of Rhizoctonia, in particular Rhizoctonia solani, a strain of Streptomyces, in particular Streptomyces scabies, or a strain of Ulocladium, in particular Ulocladium consortiale. In a most preferred embodiment the cutinase is derived from a strain of Humicola insolens, in particular the strain Humicola insolens DSM 1800. Humicola insolens cutinase is described in WO 96/13580 which is hereby incorporated by reference. The cutinase may be a variant, such as one of the variants disclosed in WO 00/34450 and WO 01/92502, which are hereby incorporated by reference. Preferred cutinase variants include variants listed in Example 2 of WO 01/92502, which is hereby specifically incorporated by reference.

Preferred commercial cutinases include NOVOZYM™ 51032 (available from Novozymes A/S, Denmark).

Additional components may also include phospholipase classified as EC 3.1.1.4 and/or EC 3.1.1.32. As used herein, the term phospholipase is an enzyme which has activity towards phospholipids. Phospholipids, such as lecithin or phosphatidylcholine, consist of glycerol esterified with two fatty acids in an outer (sn-1) and the middle (sn-2) positions and esterified with phosphoric acid in the third position; the phosphoric acid, in turn, may be esterified to an amino-alcohol. Phospholipases are enzymes which participate in the hydrolysis of phospholipids. Several types of phospholipase activity can be distinguished, including phospholipases A₁ and A₂ which hydrolyze one fatty acyl group (in the sn-1 and sn-2 position, respectively) to form lysophospholipid; and lysophospholipase (or phospholipase B) which can hydrolyze the remaining fatty acyl group in lysophospholipid. Phospholipase C and phospholipase D (phosphodiesterases) release diacyl glycerol or phosphatidic acid respectively.

The term phospholipase includes enzymes with phospholipase activity, e.g., phospholipase A (A₁ or A₂), phospholipase B activity, phospholipase C activity or phospholipase D activity. The term “phospholipase A” used herein in connection with an enzyme of the invention is intended to cover an enzyme with Phospholipase A₁ and/or Phospholipase A₂ activity. The phospholipase activity may be provided by enzymes having other activities as well, such as, e.g., a lipase with phospholipase activity. The phospholipase activity may, e.g., be from a lipase with phospholipase side activity. In other embodiments of the invention the phospholipase enzyme activity is provided by an enzyme having essentially only phospholipase activity and wherein the phospholipase enzyme activity is not a side activity.

The phospholipase may be of any origin, e.g., of animal origin (such as, e.g., mammalian), e.g. from pancreas (e.g., bovine or porcine pancreas), or snake venom or bee venom. Preferably the phospholipase may be of microbial origin, e.g., from filamentous fungi, yeast or bacteria, such as the genus or species Aspergillus, e.g., A. niger; Dictyostelium, e.g., D. discoideum; Mucor, e.g. M. javanicus, M. mucedo, M. subtilissimus; Neurospora, e.g. N. crassa; Rhizomucor, e.g., R. pusillus; Rhizopus, e.g. R. arrhizus, R. japonicus, R. stolonifer; Sclerotinia, e.g., S. libertiana; Trichophyton, e.g. T. rubrum; Whetzelinia, e.g., W. sclerotiorum; Bacillus, e.g., B. megaterium, B. subtilis; Citrobacter, e.g., C. freundii; Enterobacter, e.g., E. aerogenes, E. cloacae Edwardsiella, E. tarda; Erwinia, e.g., E. herbicola; Escherichia, e.g., E. coli; Klebsiella, e.g., K. pneumoniae; Proteus, e.g., P. vulgaris; Providencia, e.g., P. stuartii; Salmonella, e.g. S. typhimurium; Serratia, e.g., S. liquefasciens, S. marcescens; Shigella, e.g., S. flexneri; Streptomyces, e.g., S. violeceoruber; Yersinia, e.g., Y. enterocolitica. Thus, the phospholipase may be fungal, e.g., from the class Pyrenomycetes, such as the genus Fusarium, such as a strain of F. culmorum, F. heterosporum, F. solani, or a strain of F. oxysporum. The phospholipase may also be from a filamentous fungus strain within the genus Aspergillus, such as a strain of Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus niger or Aspergillus oryzae.

Preferred phospholipases are derived from a strain of Humicola, especially Humicola lanuginosa. The phospholipase may be a variant, such as one of the variants disclosed in WO 00/32758, which are hereby incorporated by reference. Preferred phospholipase variants include variants listed in Example 5 of WO 00/32758, which is hereby specifically incorporated by reference. In another preferred embodiment the phospholipase is one described in WO 04/111216, especially the variants listed in the table in Example 1.

Preferably the phospholipase is derived from a strain of Fusarium, especially Fusarium oxysporum. The phospholipase may be the one concerned in WO 98/026057 derived from Fusarium oxysporum DSM 2672, or variants thereof.

The phospholipase is preferably a phospholipase A₁ (EC. 3.1.1.32). or a phospholipase A₂ (EC.3.1.1.4.).

Examples of commercial phospholipases include LECITASE™ and LECITASE™ ULTRA, YIELSMAX, or LIPOPAN F (available from Novozymes A/S, Denmark).

Suitable amylases (alpha and/or beta) include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, alpha-amylases obtained from Bacillus, e.g. a special strain of B. licheniformis, described in more detail in GB 1,296,839, or the Bacillus sp. strains disclosed in WO 95/026397 or WO 00/060060.

Examples of useful amylases are the variants described in WO 94/02597, WO 94/18314, WO 96/23873, WO 97/43424, WO 01/066712, WO 02/010355, WO 02/031124 and PCT/DK2005/000469 (which references all incorporated by reference.

Commercially available amylases are Duramyl™, Termamyl™, Termamyl Ultra™, Natalase™, Stainzyme™, Fungamyl™ and BAN™ (Novozymes A/S), Rapidase™ and Purastar™ (from Genencor International Inc.).

Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g. the fungal cellulases produced from Humicola insolens, Thielavia terrestris, Myceliophthora thermophila, and Fusarium oxysporum disclosed in U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,648,263, U.S. Pat. No. 5,691,178, U.S. Pat. No. 5,776,757, WO 89/09259, WO 96/029397, and WO 98/012307.

Especially suitable cellulases are the alkaline or neutral cellulases having color care benefits. Examples of such cellulases are cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, U.S. Pat. No. 5,457,046, U.S. Pat. No. 5,686,593, U.S. Pat. No. 5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299. Commercially available cellulases include Celluzyme™, Carezyme™, Endolase™, Renozyme™ (Novozymes A/S), Clazinase™ and Puradax HA™ (Genencor International Inc.), and KAC-500 (B)™ (Kao Corporation).

Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g. from C. cinereas, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257. Commercially available peroxidases include Guardzyme™ and Novozym™ 51004 (Novozymes A/S).

Examples of pectate lyases include pectate lyases that have been cloned from different bacterial genera such as Erwinia, Pseudomonas, Klebsiella and Xanthomonas, as well as from Bacillus subtilis (Nasser et al. (1993) FEBS Letts. 335:319-326) and Bacillus sp. YA-14 (Kim et al. (1994) Biosci. Biotech. Biochem. 58:947-949). Purification of pectate lyases with maximum activity in the pH range of 8-10 produced by Bacillus pumilus (Dave and Vaughn (1971) J. Bacteriol. 108:166-174), B. polymyxa (Nagel and Vaughn (1961) Arch. Biochem. Biophys. 93:344-352), B. stearothermophilus (Karbassi and Vaughn (1980) Can. J. Microbiol. 26:377-384), Bacillus sp. (Hasegawa and Nagel (1966) J. Food Sci. 31:838-845) and Bacillus sp. RK9 (Kelly and Fogarty (1978) Can. J. Microbiol. 24:1164-1172) have also been described. Any of the above, as well as divalent cation-independent and/or thermostable pectate lyases, may be used in practicing the invention. In preferred embodiments, the pectate lyase comprises the amino acid sequence of a pectate lyase disclosed in Heffron et al., (1995) Mol. Plant-Microbe Interact. 8: 331-334 and Henrissat et al., (1995) Plant Physiol. 107: 963-976. Specifically contemplated pectatel lyases are disclosed in WO 99/27083 and WO 99/27084. Other specifically contemplates pectate lyases derived from Bacillus licheniformis is disclosed in U.S. Pat. No. 6,284,524 (which document is hereby incorporated by reference). Specifically contemplated pectate lyase variants are disclosed in WO 02/006442, especially the variants disclosed in the Examples in WO 02/006442 (which document is hereby incorporated by reference).

Examples of commercially available alkaline pectate lyases include BIOPREP™ and SCOURZYME™ L from Novozymes A/S, Denmark.

Examples of mannanases (EC 3.2.1.78) include mannanases of bacterial and fungal origin. In a specific embodiment the mannanase is derived from a strain of the filamentous fungus genus Aspergillus, preferably Aspergillus niger or Aspergillus aculeates (WO 94/25576). WO 93/24622 discloses a mannanase isolated from Trichoderma reseei. Mannanases have also been isolated from several bacteria, including Bacillus organisms. For example, Talbot et al., Appl. Environ. Microbiol., Vol. 56, No. 11, pp. 3505-3510 (1990) describes a beta-mannanase derived from Bacillus stearothermophilus. Mendoza et al., World J. Microbiol. Biotech., Vol. 10, No. 5, pp. 551-555 (1994) describes a beta-mannanase derived from Bacillus subtilis.

JP-A-03047076 discloses a beta-mannanase derived from Bacillus sp. JP-A-63056289 describes the production of an alkaline, thermostable beta-mannanase. JP-A-63036775 relates to the Bacillus microorganism FERM P-8856 which produces beta-mannanase and beta-mannosidase. JP-A-08051975 discloses alkaline beta-mannanases from alkalophilic Bacillus sp. AM-001. A purified mannanase from Bacillus amyloliquefaciens is disclosed in WO 97/11164. WO 91/18974 describes a hemicellulase such as a glucanase, xylanase or mannanase active. Contemplated are the alkaline family 5 and 26 mannanases derived from Bacillus agaradhaerens, Bacillus licheniformis, Bacillus halodurans, Bacillus clausii, Bacillus sp., and Humicola insolens disclosed in WO 99/64619. Especially contemplated are the Bacillus sp. mannanases concerned in the Examples in WO 99/64619 which document is hereby incorporated by reference.

Examples of commercially available mannanases include Mannaway™ available from Novozymes A/S Denmark.

Any enzyme present in a composition may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in e.g. WO 92/19709 and WO 92/19708.

The fabric wash compositions may comprise a fabric wash detergent material selected from non-soap anionic surfactant, nonionic surfactants, soap, amphoteric surfactants, zwitterionic surfactants and mixtures thereof.

Detergent compositions suitable for use in domestic or industrial automatic fabric washing machines generally contain anionic non-soap surfactant or nonionic surfactant, or combinations of the two in suitable ratio, as will be known to the person skilled in the art, optionally together with soap.

Many suitable detergent-active compounds are available and fully described in the literature, for example in “Surface-Active Agents and Detergents”, Volumes I and II, by Schwartz, Perry & Berch.

The surfactants may be present in the composition at a level of from 0.1% to 60% by weight.

Suitable anionic surfactants are well known to the person skilled in the art and include alkyl benzene sulphonate, primary and secondary alkyl sulphates, particularly C₈-C₁₅ primary alkyl sulphates; alkyl ether sulphates; olefin sulphonates; alkyl xylene sulphonates, dialkyl sulphosuccinates; ether carboxylates; isethionates; sarcosinates; fatty acid ester sulphonates and mixtures thereof. The sodium salts are generally preferred. When included therein the composition usually contains from about 1% to about 50%, preferably 10 wt %-40 wt % based on the fabric treatment composition of an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid or soap. Preferred surfactants are alkyl ether sulphates and blends of alkoxylated alkyl nonionic surfactants with either alkyl sulphonates or alkyl ether sulphates.

Preferred alkyl ether sulphates are C8-C15 alkyl and have 2-10 moles of ethoxlation. Preferred alkyl sulphates are alkylbenzene sulphonates, particularly linear alkylbenzene sulphonates having an alkyl chain length of C₈-C₁₅. The counter ion for anionic surfactants is typically sodium, although other counter-ions such as TEA or ammonium can be used. Suitable anionic surfactant materials are available in the marketplace as the ‘Genapol’™ range from Clariant.

Nonionic surfactants are also well known to the person skilled in the art and include primary and secondary alcohol ethoxylates, especially C₈-C₂₀ aliphatic alcohol ethoxylated with an average of from 1 to 20 moles of ethylene oxide per mole of alcohol, and more especially the C₁₀-C₁₅ primary and secondary aliphatic alcohols ethoxylated with an average of from 1 to 10 moles of ethylene oxide per mole of alcohol.

Non-ethoxylated nonionic surfactants include alkyl polyglycosides, glycerol monoethers and polyhydroxy amides (glucamide). Mixtures of nonionic surfactant may be used. When included therein the composition usually contains from about 0.2% to about 40%, preferably 1 to 20 wt %, more preferably 5 to 15 wt % of a non-ionic surfactant such as alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamine (“glucamides”).

Nonionic surfactants that may be used include the primary and secondary alcohol ethoxylates, especially the C₈-C₂₀ aliphatic alcohols ethoxylated with an average of from 1 to 35 moles of ethylene oxide per mole of alcohol, and more especially the C₁₀-C₁₅ primary and secondary aliphatic alcohols ethoxylated with an average of from 1 to 10 moles of ethylene oxide per mole of alcohol.

Higher levels of surfactant may be employed (up to almost 100%) but this can leave little space in the formulation for builders and other components and may lead to a sticky product which requires special processing.

Hydrotropes may be included in the composition/s. The term “hydrotrope” generally means a compound with the ability to increase the solubilities, preferably aqueous solubilities, of certain slightly soluble organic compounds. Examples of hydrotropes include sodium xylene sulfonate, SCM.

The composition/s may comprise a solvent such as water or an organic solvent such as isopropyl alcohol or glycol ethers. Solvents are typically present in liquid or gel compositions.

The composition/s may contain a metal chelating agent such as carbonates, bicarbonates, and sesquicarbonates. The metal chelating agent can be a bleach stabiliser (i.e. heavy metal sequestrant). Suitable bleach stabilisers include ethylenediamine tetraacetate (EDTA), diethylenetriamine pentaacetate (DTPA), ethylenediamine disuccinate (EDDS), and the polyphosphonates such as the Dequests (Trade Mark), ethylenediamine tetramethylene phosphonate (EDTMP) and diethylenetriamine pentamethylene phosphate (DETPMP). In general metal chelating agents will not be present in the part (a) of the composition as microbial function may be impaired if metal ions are made unavailable.

Builder materials may be selected from 1) calcium sequestrant materials, 2) precipitating materials, 3) calcium ion-exchange materials and 4) mixtures thereof.

Examples of calcium sequestrant builder materials include alkali metal polyphosphates, such as sodium tripolyphosphate and organic sequestrants, such as ethylene diamine tetra-acetic acid.

Examples of precipitating builder materials include sodium orthophosphate and sodium carbonate.

Examples of calcium ion-exchange builder materials include the various types of water-insoluble crystalline or amorphous aluminosilicates, of which zeolites are the best known representatives, e.g. zeolite A, zeolite B (also known as zeolite P), zeolite C, zeolite X, zeolite Y and also the zeolite P-type as described in EP-A-0,384,070.

The composition/s may also contain 0-65% of a builder or complexing agent such as ethylenediaminetetraacetic acid, diethylenetriamine-pentaacetic acid, alkyl- or alkenylsuccinic acid, nitrilotriacetic acid or the other builders mentioned below. Many builders are bleach-stabilising agents by virtue of their ability to complex metal ions.

Where builder is present, the composition/s may suitably contain less than 20% wt, preferably less than 10% by weight, and most preferably less than 10% wt of detergency builder.

The composition/s may contain as builder a crystalline aluminosilicate, preferably an alkali metal aluminosilicate, more preferably a sodium aluminosilicate. This is typically present at a level of less than 15% w. Aluminosilicates are materials having the general formula:

0.8-1.5M₂O.Al₂O₃.0.8-6SiO₂

where M is a monovalent cation, preferably sodium. These materials contain some bound water and are required to have a calcium ion exchange capacity of at least 50 mg CaO/g. The preferred sodium aluminosilicates contain 1.5-3.5 SiO₂ units in the formula above. They can be prepared readily by reaction between sodium silicate and sodium aluminate, as amply described in the literature. The ratio of surfactants to alumuminosilicate (where present) is preferably greater than 5:2, more preferably greater than 3:1.

Alternatively, or additionally to the aluminosilicate builders, phosphate builders may be used. In this art the term ‘phosphate’ embraces diphosphate, triphosphate, and phosphonate species. Other forms of builder include silicates, such as soluble silicates, metasilicates, layered silicates (e.g. SKS-6 from Hoechst).

For low cost formulations carbonate (including bicarbonate and sesquicarbonate) and/or citrate may be employed as builders.

The composition may comprise one or more polymers. Examples are carboxymethylcellulose, poly(vinylpyrrolidone), poly (ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.

Modern detergent compositions typically employ polymers as so-called ‘dye-transfer inhibitors’. These prevent migration of dyes, especially during long soak times. Any suitable dye-transfer inhibition agents may be used in accordance with the present invention. Generally, such dye-transfer inhibiting agents include polyvinyl pyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, manganese pthalocyanine, peroxidases, and mixtures thereof.

Nitrogen-containing, dye binding, DTI polymers are preferred. Of these polymers and co-polymers of cyclic amines such as vinyl pyrrolidone, and/or vinyl imidazole are preferred.

Polyamine N-oxide polymers suitable for use herein contain units having the following structural formula: R-A_(x)-P; wherein P is a polymerizable unit to which an N—O group can be attached or the N—O group can form part of the polymerizable unit; A is one of the following structures: —NC(O)—, —C(O)O—, —S—, —O—, —N═; x is 0 or 1; and R is an aliphatic, ethoxylated aliphatic, aromatic, heterocyclic or alicyclic group or combination thereof to which the nitrogen of the N—O group can be attached or the N—O group is part of these groups, or the N—O group can be attached to both units. Preferred polyamine N-oxides are those wherein R is a heterocyclic group such as pyridine, pyrrole, imidazole, pyrrolidine, piperidine and derivatives thereof. The N—O group can be represented by the following general structures: N(O)(R)₀₋₃, or ═N(O)(R′)₀₋₁, wherein each R′ independently represents an aliphatic, aromatic, heterocyclic or alicylic group or combination thereof; and the nitrogen of the N—O group can be attached or form part of any of the aforementioned groups. The amine oxide unit of the polyamine N-oxides has a pKa<10, preferably pKa<7, more preferably pKa<6.

Any polymer backbone can be used provided the amine oxide polymer formed is water-soluble and has dye transfer inhibiting properties. Examples of suitable polymeric backbones are polyvinyls, polyalkylenes, polyesters, polyethers, polyamides, polyimides, polyacrylates and mixtures thereof. These polymers include random or block copolymers where one monomer type is an amine N-oxide and the other monomer type is an N-oxide. The amine N-oxide polymers typically have a ratio of amine to the amine N-oxide of 10:1 to 1:1,000,000. However, the number of amine oxide groups present in the polyamine oxide polymer can be varied by appropriate copolymerization or by an appropriate degree of N-oxidation. The polyamine oxides can be obtained in almost any degree of polymerization. Typically, the average molecular weight is within the range of 500 to 1,000,000; more preferably 1,000 to 500,000; most preferably 5,000 to 100,000. This preferred class of materials is referred to herein as “PVNO”. A preferred polyamine N-oxide is poly(4-vinylpyridine-N-oxide) which as an average molecular weight of about 50,000 and an amine to amine N-oxide ratio of about 1:4.

Copolymers of N-vinylpyrrolidone and N-vinylimidazole polymers (as a class, referred to as “PVPVI”) are also preferred. Preferably the PVPVI has an average molecular weight range from 5,000 to 1,000,000, more preferably from 5,000 to 200,000, and most preferably from 10,000 to 20,000, as determined by light scattering as described in Barth, et al., Chemical Analysis, Vol. 113. “Modern Methods of Polymer Characterization”. The preferred PVPVI copolymers typically have a molar ratio of N-vinylimidazole to N-vinylpyrrolidone from 1:1 to 0.2:1, more preferably from 0.8:1 to 0.3:1, most preferably from 0.6:1 to 0.4:1. These copolymers can be either linear or branched. Suitable PVPVI polymers include Sokalan™ HP56, available commercially from BASF, Ludwigshafen, Germany.

Also preferred as dye transfer inhibition agents are polyvinylpyrrolidone polymers (“PVP”) having an average molecular weight of from about 5,000 to about 400,000, preferably from about 5,000 to about 2000,000, and more preferably from about 5,000 to about 50,000. PVP's are disclosed for example in EP-A-262,897 and EP-A-256,696. Suitable PVP polymers include Sokalan™ HP50, available commercially from BASF. Compositions containing PVP can also contain polyethylene glycol (“PEG”) having an average molecular weight from about 500 to about 100,000, preferably from about 1,000 to about 10,000. Preferably, the ratio of PEG to PVP on a ppm basis delivered in wash solutions is from about 2:1 to about 50:1, and more preferably from about 3:1 to about 10:1.

Also suitable as dye transfer inhibiting agents are those from the class of modified polyethyleneimine polymers, as disclosed for example in WO-A-0005334. These modified polyethyleneimine polymers are water-soluble or dispersible, modified polyamines. Modified polyamines are further disclosed in U.S. Pat. No. 4,548,744; U.S. Pat. No. 4,597,898; U.S. Pat. No. 4,877,896; U.S. Pat. No. 4,891,160; U.S. Pat. No. 4,976,879; U.S. Pat. No. 5,415,807; GB-A-1,537,288; GB-A-1,498,520; DE-A-28 29022; and JP-A-06313271.

Preferably the composition/s according to the present invention comprises a dye transfer inhibition agent selected from polyvinylpyrridine N-oxide (PVNO), polyvinyl pyrrolidone (PVP), polyvinyl imidazole, N-vinylpyrrolidone and N-vinylimidazole copolymers (PVPVI), copolymers thereof, and mixtures thereof.

The amount of dye transfer inhibition agent in the composition/s according to the present invention will be from 0.01 to 10%, preferably from 0.02 to 5%, more preferably from 0.03 to 2%, by weight of the composition.

The composition/s may also contain other detergent ingredients such as e.g. fabric conditioners including clays, foam boosters, suds suppressors (anti-foams), anti-corrosion agents, soil-suspending agents, anti-soil redeposition agents, further dyes, anti-microbials, optical brighteners, tarnish inhibitors, or perfumes.

The dispensing drawer of an automatic washing machine may be used to sequentially dose the enzymes, for example by using the pre-wash chamber to dose the protease/s and the main wash chamber to dose the lipase/s.

The invention may also be employed in hand washing operation, which can be effected without the need for additional devices, but enzymes are simply added sequentially to the wash liquor in which the items are being washed.

A hand washing operation may however involve wash tools such as scrubbing devices and the compositions may be applied directly to said tools, in sequence in accordance with the invention, and the tools then used to clean the clothes.

The tools or devices may also be impregnated and/or may function as a delivery device to deliver said enzymes in a washing/stain removal (preferably grass stain removal) process according to the invention.

EXAMPLES OF NON LIMITING EMBODIMENTS OF THE INVENTION Example 1 Wash Evaluation of Protease and a Lipase in Grass Stain Removal (Tergotometer)

Wash performance was evaluated by washing grass stained polyester swatches (wfk30A) in a detergent solution with a protease (Savinase 12TXT) and a lipase (Lipex 100T or lipolase 100T) in single enzyme, combination and sequence (2 wash) treatments.

Preparation of Grass Stain Swatches: Consumer Relevant stains were prepared manually by rigorously rubbing clumps of lawn grass on clean cotton fabric swatches, to create circular homogeneous dark green stains.

Wash: Grass stain swatches (7×7 cm) were placed in tergotometer tubs with the model formulation components (table 1) in 11 demin water and incubated for 30 min at 37° C., with mechanical stirring. At t=15 minutes, stirring was paused, all stains were rinsed in 11 demin water, and stains were transferred to second-in-sequence enzyme wash tubs. Stirring was continued for the further 15 minutes. The swatches were rinsed in tap water, span and allowed to dry in the dark, at room temperature overnight.

Evaluation: Colour remission of the swatches was measured at 410 nm using a Hunterlab UltraScan VIS remission spectrophotometer. The results are expressed as delta remission=(R_(after wash)−R_(before wash))_(enzyme)−(R_(after wash)−R_(before wash))_(control), where R is the remission at 410 nm using the CIE L*a*b* (CIELAB) values generated (FIG. 1).

TABLE 1 Formulation components NaCl 0.05 M Ca²⁺ 6 FH CAPS buffer, pH 10 20 mM Surfactant (80:20 0.5 g/l LAS:EO7, w/w) Enzyme (total) 1 mg/L

TABLE 2 Wash performance of savinase, savinase and lipase and savinase followed by lipase on cotton and at low FH (FH6) in tergotometer. All stains are washed with surfactant. Total enzyme protein concentration = 1 μg/ml. Enzyme d[DE]minus control Savinase 0.82 Savinase + d Lipex 0.68 Savinase + Lipolase 1.61 Savinase followed by 2.81 Lipex Savinase the Lipolase 3.46

The table shows how the sequential dosing of a protease followed by a lipase provides much improved stain removal.

Example 2 Wash Evaluation of Protease and a Lipase in Grass Stain Removal (Microtiter)

Wash performance was evaluated by washing grass stained cotton testcloth in a detergent solution with a protease (Savinase 12TXT) and a lipase (Lipex 100T) in single enzyme, combination and sequence (2 wash) treatments.

Preparation of Grass Stain Testcloth: Consumer Relevant stains were prepared manually by rigorously rubbing blended grass (3:1 ration in water), prefiltered through a polyester fabric, on clean cotton fabric using a nailbrush.

Wash: Grass stain testcloths were placed in microtiter plates with the model formulation components (table 1) in 200 μl demin water and incubated for 30 min at 37° C. on an orbital shaker, with agitation (1150 rpm). This time low (FH60) and high (FH40) water hardness was tested. At t=15 minutes, agitation was paused, all stains were rinsed in 200 μl demin water, and exposed to second-in-sequence enzyme solution. Agitation was continued for the further 15 minutes. The swatches were rinsed in tap water twice and allowed to dry in the dark, at 45° C. for at least 3 hours.

Evaluation: Colour remission of the swatches was measured at 410 nm using a flatbed remission spectrophotometer. The results are expressed as delta remission ═(R_(after wash)−R_(before wash))_(enzyme)−(R_(after wash)−R_(before wash))_(control), where R is the remission at 410 nm using the CIE L*a*b* (CIELAB) values generated (tables 3 and 4).

TABLE 3 Wash performance of savinase, savinase and lipase and savinase followed by lipase on cotton and at low FH (FH6) in microtiter plates. All stains are washed with surfactant. Total enzyme protein concentration = 1 or 40 μg/ml. S-L, savinase followed by lipase. Savinase 15.39, 16.12, Savinase + Lipase 14.17 17.95, Savinase followed by 20.40 21.10 Lipase

TABLE 4 Wash performance of savinase, savinase and lipase and savinase followed by lipase on cotton and at high FH (FH40) in microtiter plates. All stains are washed with surfactant. Total enzyme protein concentration = 1 or 40 μg/ml. S-L, savinase followed by lipase. Savinase 6.66 12.19 Savinase + Lipase 6.52 10.72, Savinase followed by 8.90 13.94 Lipase

It is of course to be understood that the invention is not intended to be restricted to the details of the above embodiment which are described by way of example only. 

1. A washing machine incorporating a sequential treatment device for sequentially treating fabrics with at least first and second enzymes, the device comprising a plurality of separate chambers containing respectively first and second enzymes, from which chambers the enzymes are sequentially dispensed characterised in that the first enzyme(s) comprise a protease and the second enzyme(s) comprise one or more enzymes of a different family to the first enzyme.
 2. A washing machine according to claim 1 characterised in that the second enzyme comprises one or more lipolytic enzymes.
 3. The washing machine of claim 1 wherein the sequential treatment device comprises the drawer of a washing machine. 